The immediate goals of this program are to elucidate how mutations in NOTCH1 receptors cause aberrant increases in signaling, and how these increases in signaling contribute to the pathogenesis of human T-cell acute lymhoblastic lymphoma/leukemia (T-ALL). Guided by these mechanistic insights, the long-term goal is to develop novel small molecule and antibody reagents that will afford new diagnostic and rational therapeutic approaches in T-ALL and other malignancies associated with increased NOTCH signaling. Project 1 will i), use cell-based assays to determine how mutations in the ectodomain and PEST domain of NOTCH1 cause increased production and stability of activated NOTCH1, respectively, and thereby synergize to increase NOTCH1 signals;ii), use mouse models to determine how furin cleavage, NOTCH ligands, and specific C-terminal residues contribute to the development of leukemia;and iii), determine how leukemia associated mutated NOTCH1 receptors of varying strength influence human hematopoietic stem cell differentiation and transformation. Project 2 will use a variety of mouse models and cell lines to: i) determine the leukemogenic potential of various NOTCH1 ectodomain and PEST mutations;ii) identify the cell of origin for NOTCH1 leukemia and study the interaction of NOTCH1 with other proteins implicated in human T-ALL;and iii) identify critical target genes downstream of activated NOTCH1 that contribute to its leukemogenecity. Project 3 will determine the structures of the NOTCH1 heterodimerization domain and the core NOTCH1 transcriptional activation complex, study the effects of mutations on the structure and stability of the purified heterodimerization domain, and conduct small molecule screens to identify inhibitors of the nuclear NOTCH1 transcriptional activation complex. The activities of this highly focused program will be organized by an Administrative Core, and supported by a Protein Core, which will make proteins for functional studies and antibody production. The proposed studies and activities of this program will yield critical mechanistic insights into normal and leukemogenic NOTCH1 signaling, and produce valuable reagents for the scientific community as a whole, thereby creating new translational opportunities in cancers associated with altered NOTCH signaling.